1. Field of the Invention
The present invention generally relates to plant genetics. More specifically, the invention relates to methods and compositions for the modulation of flavonoid and isoflavonoid biosynthesis in plants.
2. Description of the Related Art
Isoflavonoid natural products are limited primarily to leguminous plants, where they function as pre-formed or inducible antimicrobial or anti-insect compounds, as inducers of the nodulation genes of symbiotic Rhizobium bacteria, and as allelopathic agents (Dixon, 1999). However, much attention is now being focused on the simple isoflavones daidzein and genistein in view of many reports linking their dietary consumption to a range of potential health benefits (Barnes, 1998; Barnes, 1996; Adlercreutz. and Mazur, 1997; Adlercreutz, 1998; Dixon, 1999; Dixon, 2002). The major dietary sources of these compounds for humans are soybean seed products, which are rich in daidzein and genistein, and chickpea seeds, which also contain biochanin A (4′-O-methylgenistein). The structures of these isoflavones are shown in FIG. 1.
Epidemiological studies have demonstrated a link between consumption of soy isoflavones and reduced risks of breast and prostate cancers in humans (Messina et al., 1994; Adlercreutz, 1998). Genistein and biochanin A exhibit chemopreventative activity against chemically-induced cancers in a number of mammalian cell model systems (Fotsis et al., 1995; Lamartiniere et al., 1995; Rauth et al, 1997; Uckun et al., 1995; Yanagihara et al., 1993). Furthermore, isoflavones have been shown to possess other health promoting activities, including potential chemoprevention of osteoporosis and cardiovascular disease (Anderson and Garner, 1998; Draper et al., 1997; Tikkanen et al., 1998; Wagner et al., 1997). In addition to isoflavonoids, many other flavonoid-derived compounds have been ascribed health-promoting activity. These include flavonols, such as quercetin, that occur at significant levels in leaves and fruit of many plant species and that have high antioxidant activity (Rice-Evans and Miller, 1996).
Isoflavonoids are formed by a branch of the flavonoid biosynthetic pathway, and originate from a central flavanone intermediate that is ubiquitously present in plants. For entry into the isoflavonoid pathway, the flavanone naringenin undergoes migration of the B-ring from the 2- to the 3-position followed by hydroxylation at the 2-position catalyzed by a microsomal cytochrome P450 enzyme, CYP93C1 (2-hydroxyisoflavanone synthase or 2-HIS, also commonly termed isoflavone synthase (IFS) (FIG. 1). The resulting 2-hydroxyisoflavanone then undergoes dehydration to yield the corresponding isoflavone genistein (Hakamatsuka et al., 1990; Kochs and Grisebach, 1986), as shown in FIG. 1. The dehydration reaction can take place non-enzymatically in vitro under acid conditions (Kochs and Grisebach, 1986), although an enzyme has been purified from cell cultures of Pueraria lobata that can catalyze this reaction (Hakamatsuka et al., 1998). This dehydratase appears to be closely associated with the microsomal aryl migration enzyme (Hakamatsuka et al., 1998).
cDNA clones that encode IFS have been characterized from soybean and other legumes (Akashi et al., 1999; Jung et al., 2000; Steele et al., 1999). The soybean enzyme is classified as CYP93C1v2. When expressed in insect cells, it converts the flavanones liquiritigenin and naringenin directly to their corresponding isoflavones daidzein and genistein in the presence of NADPH (Steele et al., 1999), as shown in FIG. 1. It is not clear whether dehydration of the putative 2-hydroxyisoflavanone intermediate occurs on the enzyme, or results from an endogenous dehydratase activity present in the insect cell microsomes. The aryl migration enzyme from licorice (Glycyrrhiza echinata) has been shown to produce a 2-hydroxyisoflavanone from a flavanone when expressed in yeast (Akashi et al., 1999).
There have been few reports to date on the introduction of new natural product pathways into plants through genetic manipulation. An important recent example is the introduction of the provitamin A pathway into rice, a process that involved the introduction of three genes (Ye et al., 2000). The soybean IFS has been introduced into the model crucifer Arabidopsis thaliana by Agrobacterium-mediated transformation (Jung et al., 2000; International Application No. PCT/US00/05915, filed Mar. 8, 2000) and into tobacco and corn by Agrobacterium or biolistic bombardment methods, respectively (Yu et al., 2000). These studies have shown the production of low levels of genistein (up to a maximum of around 40 nmol/g fresh weight, but generally less) after hydrolysis of potential genistein glyco-conjugates that may have been formed in the transgenic plants. The nature of the potential glyco-conjugates has not been determined. While these studies confirm production of isoflavones in transgenic plants, it would be advantageous to increase the amount of flavonoid natural products produced by the transgenic plants to provide improved bioactivity for dietary health-promotion.
The key biosynthetic reactions leading to the formation of flavanone are catalyzed by chalcone synthase (CHS) and chalcone isomerase (CHI). CHS is a homodimeric polyketide synthase that forms 2′,4,′4′,6′-tetrahydroxychalcone (naringenin chalcone) from three molecules of malonyl coenzyme A and one molecule of 4-coumaroyl CoA according to the reaction shown in FIG. 1. It is often encoded by a multigene family in legumes (Junghans et al., 1993), and many CHS genes have been cloned from a wide number of plant species, including alfalfa (Schröder, 1997).
CHI is a monomeric enzyme that very efficiently catalyzes the isomerization of naringenin chalcone to its corresponding flavanone, naringenin (4′,5,7-trihydroxy-flavanone), as depicted in FIG. 1. This reaction can also occur spontaneously at alkaline pH, although without a stereochemical direction (CHI specifically catalyzes formation of (−) flavanone). For this reason, few plants accumulate naringenin chalcone. CHI from certain legumes can also act on 2′,4,4′-trihydroxychalcone (isoliquiritigenin), to form the corresponding flavanone liquiritigenin (4′, 7, dihydroxy-flavanone) (Dixon et al., 1988), whereas the enzyme from most non-legumes does not appear to have this activity. CHI genes have now been cloned from several species, including alfalfa (McKhann and Hirsch, 1994). In view of its high activity level in many plant tissues relative to the activity level of CHS, CHI has not heretofore been thought of as a rate-determining enzyme for flavanone formation. However, it has recently been shown that CHI is rate limiting for flavonoid biosynthesis in tomato fruit peel (Muir et al., 2001).
However, in that particular study, expression of the 35S promoter driven petunia CHI transgene did not increase flavonol levels in the flesh of the fruit, or in the leaves. The peel of wild-type tomato fruit unusually contains high levels of naringenin chalcone but very low levels of flavonols, suggesting limitation of CHI activity and explaining the approximately 80-fold increase in flavonols following expression of the CHI transgene (Muir et al., 2001). Such an increase was not observed when maize CHI was over-expressed in wild-type Arabidopsis and, in fact, such plants appeared to have reduced levels of anthocyanins (Dong et al., 2001).
The foregoing studies have provided a further understanding of the metabolism of plant secondary metabolism. However, the prior art has failed to provide techniques for the application of this understanding to the creation of plants having valuable new characteristics. What are thus needed are practical techniques for the production of novel plants with improved phenotypes and methods for the use thereof. Such techniques may allow the creation and use of plants with improved nutritional quality, thereby benefiting both human and animal health and representing a substantial benefit in the art.